Spheroids have become an essential tool in preclinical cancer research. The uniformity of spheroids is a critical parameter in drug test results. Spheroids form by self-assembly of cells. Hence, the control of homogeneity of spheroids in terms of size, shape, and density is challenging. We developed surface-optimized polydimethylsiloxane (PDMS) biochip platforms for uniform spheroid formation on-chip. These biochips were surface modified with 10% bovine serum albumin (BSA) to effectively suppress cell adhesion on the PDMS surface. These surface-optimized platforms facilitate cell self-aggregations to produce homogenous non-scaffold-based spheroids. We produced uniform spheroids on these biochips using six different established human cell lines and a co-culture model. Here, we observe that the concentration of the BSA is important in blocking cell adhesion to the PDMS surfaces. Biochips treated with 3% BSA demonstrated cell repellent properties similar to the bare PDMS surfaces. This work highlights the importance of surface modification on spheroid production on PDMS-based microfluidic devices. Cancer is among the top causes of death in developed countries. According to the statistics in Canada , cancer Focusing on spheroids, various techniques have been used for their formation, with the same principle, whereby adhesive forces between cells are more prominent than cell–substrate adhesion ,7. ProduWe previously designed a PDMS-based tumor-on-a-chip model for capturing tumor cells and generating uniform spheroids . We obseThe general design of the channel is similar to our previous work, which was adapted from the design developed by Astolfi et al. . In this®, Mann Formulated Products, Macungie, PA, USA) prior to replication of PDMS. To make PDMS replicas, as described before [To obtain microfluidic devices from the 3D-printed mold, we made PDMS replicas by soft lithography. To prevent sticking of the PDMS to the resin mold, we pre-treated molds with a silicone spray  were prepared [Before introducing the cells to the devices, by using 99.9% ethanol , microfluidic channels were sterilized and the air bubbles were removed from the channels. To reduce cell adhesion to the PDMS surfaces and promote the spheroid formation, we performed surface coating with BSA. BSA (Sigma-Aldrich) solutions in two different concentrations of 3% prepared  and ster2, 95% ambient air) right after the cell seeding process.Then, by incubation of freshly prepared BSA solutions with biochips, their surfaces were modified to assess spheroid formation on-chip. One group of biochips was surface treated with 3% BSA, and another group of biochips was surface treated with 10% BSA to assess the impact of surface treatment on spheroid production. Only 10% BSA was used for co-culture model assays. All the devices were incubated overnight in the incubator , stromal connective tissue cells including IRM-90 mCherry Fibroblasts , derivative subline of human prostate cancer LNCaP-derived C4-2B cells, human non-small lung adenocarcinoma cell line H1299, human lung carcinoma epithelial cell line A549, and human glioblastoma cell line U251. MDA-MB231, IRM-90, and C4-2B were kindly provided by the laboratory of Prof. M. Park at McGill University. H1299 and A549 were generously provided by the laboratory of Prof. M. Lavertu at Polytechnique Montreal. U251cell line was a generous gift from the laboratory of Dr. R. J. Diaz at The Montreal Neurological Institute-Hospital, McGill University. C4-2B, H1299, A549, and U251 cells were fluorescently labeled with cell-labeling solution  in accordance with the manufacturer’s protocol.v/v fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin (PS). All the other cell lines were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented as above . All the cell lines (less than 5 passages) were seeded in T-75 flasks  at the density of 1.5 × 106 cells per flask and placed in the incubator at 37 °C, 5% CO2 and 95% relative humidity (RH). After reaching 80–90% confluence, cells were washed two times with sterile phosphate buffered saline , and trypsinized with 0.25% Trypsin-EDTA solution . Then supplemented fresh culture medium  was added to the cell suspensions and centrifuged for 5 min at 1500 rpm to collect the cells. Prior to cell seeding, microfluidic channels were rinsed properly with sterile PBS  three times to clean the channels from the BSA residues, then the fluid within the channels was replaced with a fresh supplemented culture medium. Then, cell suspensions with the concentration of 1 × 106 cells/mL were injected into the microfluidic channels inlets by using a micropipette (P200) and at the same time, 100 µL of the medium were removed from the channel’s outlet. Consequently, the cell suspension was pipetted into the outlet, and 100 µL of cell suspension inside the channel was removed from the inlet. To provide uniform and homogenous cell distribution across the channels, we repeated this process on both sides (inlet and outlet) of the channels three times. Following the cell seeding process, microfluidic devices were put in the incubator. The culture medium was refreshed every 24 h using a micropipette (P200).H1299 and C4-2B cells were cultured in RPMI-1640 medium supplemented with 10% Using Epifluorescence inverted microscope  and sCMOS camera  and the objective lens EC Plan-Neofluar 5×/0.15, spheroid formation and proliferation were imaged directly through the thin PDMS layer for the duration of 7 days. The size of the spheroids, their compactness, and their spherical geometry was quantitated on day 1, 3, and 7 using Image J software.The images of the spheroids that were recorded as explained above were used for analysis with Image J  to assess spheroid growth and proliferation. All images had a high resolution of more than 1900 dpi. The brightness level of fluorescent images was assessed by a digit value from zero to a maximum of 255. The average mean diameter of each spheroid can be written as D m = (D max + D min)/2 from 10 repeats. We reported the average size of the spheroids as mean diameter ± standard deviation. We defined the circularity of the spheroids by the Circularity = 100 × (D min/D max around a single sphere). Brightness level ratio is equal to BLR = (Brightness level/255) × 100. We used fluorescent signals as the function of cell proliferation in the spheroids. All Data have been reported as the mean ± SE of minimum of three independent tests. All error bars in figures indicate SE. One representative experiment is presented where the same trends were seen in multiple trials.6 cells/mL in all the experiments. Process of cell seeding and spheroid formation on-chip was repeated as described above for mono-type MCTSs.We assessed spheroid morphology and proliferation in co-culture models. 3D tumor model consisting of a co-culture of A549 cells and IRM-90 cells  have been used to form hetero-type MCTSs in comparison with a monoculture of either A549 or IRM-90. The initial cell suspension had a concentration of 1 × 10As a comparison and a control model for spheroid formation assays on PDMS biochips that were surface treated with 10% BSA, spheroid formation and cell adhesion were assessed on biochip treated with 3% BSA.n = 3) in order to evaluate the reproducibility of the data. For each experiment, a minimum of 10 spheroids was analyzed per condition. Statistical analyses were performed using Microsoft Excel 2016 . All data are represented as mean ± standard error of the mean. One-way ANOVA was performed to assess statistical significance between means, with post hoc Tukey tests for comparison between means. A p ≤ 0.05 was considered statistically significant.All experiments were repeated in three independent biological replicates , proliferation and spheroid compaction were observed for all the cell lines and co-culture models, except for C4-2B cells. For C4-2B cells, spherical cell agglomerations were observed on day 3 (72 h) post-seeding. All the cell lines were successfully made spheroid on biochips pre-coated with 10% BSA and spheroid growth was assessed over a period of seven days .6 cells/mL) in all groups. As a control model, we studied the spheroid formation on biochips pre-coated with 3% BSA  modulate spheroid size  on the sHere, we observed that, contrary to 2D cultures, which grow exponentially, spheroids were characterized by an exponential growth phase followed by a decrease in proliferation with an increase in non-proliferation rate. According to our observations, the duration of each phase was dependent on the type of the cell line.Spheroids can also be formed from a co-culture of tumor cells with stromal cells ,3. These6 cells/mL in each experiment.We developed scaffold-free multicellular spheroids consisting of tumor cells (A549) and fibroblast (IRM-90) to provide the crosstalk between tumor and stroma cells. We studied the effect of changes in the ratio of tumor cells and stromal cells on the size and growth pattern of the co-culture models. In terms of spheroid size, circular shape, and the chance of spheroid formation on-chip, no significant changes were observed when changing the ratio between the tumor and stromal cells in this study .According to the data presented in p < 0.0001). However, the circularity of the mono-type and hetero-type MCTSs did not show a statistically significant difference. Moreover, the chance of spheroid formation on the device by adding the stromal cells into the tumor microenvironment significantly increased (** p < 0.001)  . We consIt is proven that the presence of the stromal cells in the tumor microenvironment increases the physiological relevance of the spheroid models and influences the cellular responses to therapeutic agents ,7. Our oSpheroid size and morphology influence the robustness of in vitro assays . TherefoThe microenvironment, including multicellularity, can drastically influence the formation of spheroids ,38. RecaHere, we conclude that surface treatment with 10% BSA is a simple, effective, and reproducible method for surface modification of PDMS-based biochips to promote the formation of scaffold-free spheroid models on-chip for various cell lines studied in this work. This platform holds the possibility to be used as a tool for the assessment of the efficacy of various therapeutic strategies in vitro. A fundamental understanding of interactions between cell-surface is particularly important for directed control of cell function for biomedical applications. Future work will explore capturing patient-derived circulating tumor cells from liquid biopsies, as well as assess the impact of spheroid formation from patient-derived solid-tumor and stromal cells. The continued research effort in the field of biomaterials and surface engineering in microfluidic platforms will help to create desirable surfaces for lab-on-a-chip and organ-on-a-chip applications.


Ovarian cancer (OC) is one of the most common types of cancer among malignancies of the female reproductive system. This pathology is asymptomatic until advanced stages and has a poor prognosis. Our study aimed to search for lncRNA–miRNA–mRNA competing triplets that promote ovarian tumorigenesis. For this purpose, we analyzed tumor samples from the TCGA database and verified the results experimentally in a set of 46 paired samples of tumor and matched histologically unchanged ovarian tissues from OC patients. The list of RNAs selected in silico for experimental studies included 13 mRNAs, 10 lncRNAs, and 5 miRNAs related to epithelial–mesenchymal transition and angiogenesis. We evaluated the expression of these RNAs by qRT-PCR and assessed the correlation between levels of miRNAs, mRNAs, and lncRNAs. Sixteen significant triplets were revealed, in some of which, e.g., OIP5-AS1–miR-203a–c-MET and OIP5-AS1–miR-203a–ZEB2, both lncRNA and mRNA had sites for miR-203a direct binding. Transfection of the OVCAR-3 and SKOV-3 cell lines with the miR-203a mimic was used to confirm the novel links of miR-203a with ZEB2 and c-MET in OC. These connections suggest that the interactomes have the potential for diagnostics of metastasis at early onset. Ovarian cancer (OC) is a leading cause of death among gynecological cancers in women around the world. In Russia, the incidence of ovarian cancer is approximately 77 cases per 100 thousand population . The fivIn recent years, molecular alterations in cancer at an epigenetic level have been attracting more attention. Epigenetic regulation during tumorigenesis involves multiple steps, where DNA methylation and noncoding RNAs (ncRNAs) are the key players ,9,10,11.MIR-124-2, -125B-1, -137, -203A, and -375) showed statistically significant association with metastasis. Thereby, these miRNAs were additionally validated on a set of 13 primary tumors and matched peritoneal metastases. The obtained data evidenced that the miRNAs have potential as prediction biomarkers for OC dissemination.Indeed, in a previous study, we demonstrated that the aberrant methylation led to significant decrease in expression of 12 tumor-suppressive microRNAs (miRNAs): miR-124-3p, -125b-5p, -127-5p, -129-5p, -132-3p, -137, -148a-3p, -191-5p, -193a-5p, -203a, -339-3p, and -375 [In this study, we investigated related long noncoding RNAs (lncRNAs) and mRNAs and analyzed the shift in the lncRNA–miRNA–mRNA interactome. The fact is that noncoding RNAs such as miRNAs and lncRNAs affect the posttranscriptional level of protein-coding genes via the formation of competing endogenous RNA (ceRNA) complexes ,15,16,17In the present study, we screened expression profiles of OC samples presented in the TCGA database. Genes that were differentially expressed (DE) in ovarian tumor samples compared to normal samples were identified. Next, correlations between levels of three types of RNAs in pairs miRNA–lncRNA, miRNA–mRNA, and lncRNA–mRNA were calculated. The significant triplets were studied experimentally. By qRT-PCR, we evaluated expression profiles of the in silico selected triplets in tumor samples and matched normal tissues from patients with OC and estimated correlations between levels of lncRNAs, miRNAs, and mRNAs indicating their crosstalk. Moreover, transfection of the OVCAR-3 and SKOV-3 cell lines was performed to confirm the interactions.https://portal.gdc.cancer.gov/projects/TCGA-OV, accessed on 27 May 2021). Among them, we selected 311 cases with primary tumors and without any treatment before biopsy sampling. Most patients (72%) in the selected subgroup had stage III OC with metastasis, which is consistent with the characteristics of our sample set .p (p.adj) < 0.05). The list of DE lncRNAs is presented in In total, 354 lncRNAs were DE  confirmed the leading role of the transcriptional misregulation process in OC patients . The DE The analysis revealed 102 statistically significant correlations between miRNA and DE lncRNA levels , while fIt appeared that the expression of many DE lncRNAs in the formed pairs was not detected in a part of the samples. Thus, we added a threshold for expression presence, which was set at >0 in more than 70% of samples. After this, we eliminated pairs in which the lncRNA expression did not meet this criterion. The same cutoff was applied for mRNAs.As a result of this screening, we selected 13 mRNAs, 10 lncRNAs, and 5 miRNAs for experimental validation . Of the The qRT-PCR analysis of levels of 5 miRNAs in a set of 46 paired  ovarian tissue samples showed their downregulation in OC . HoweverNot all experimentally obtained values of lncRNA expression in our sample set were consistent with ones in the TCGA-OV . Namely,In the same way, we noticed a difference between expression values based on the TCGA data analysis and the obtained experimental results for mRNAs . WNT4 haThe calculation of Spearman’s coefficient for three types of pairs  evidenced that expression levels of all five selected tumor-suppressive miRNAs were negatively correlated with those of the mRNAs and lncRNAs.Altogether, we identified 16 triplets with significant correlation coefficients between expression levels of RNAs . Based oWe revealed only one triplet with miR-148a, which was moderately connected with MALAT1 and ADAM9 . LncRNA–Fifteen triplets were formed in conjunction with miR-203a . In all However, the analysis of the localization of miR-203 binding sites showed that binding is possible only with two out of four correlated lncRNAs (OIP5-AS1 and MLK7-AS1), and that only c-MET, ZEB1, and ZEB2 had a complementary sequence with miR-203 among six correlated mRNAs. MiR-203 had more common nucleotides in the seed site with c-MET and ZEB2 sequences .Affymetrix Human Transcriptome analysis of additional clinical samples revealed 512 DE genes, including 107 lncRNAs and 405 mRNAs. Of these, 121 DE genes were common for in silico TCGA-OV and in vitro analyses . The treTo validate possible interactions between selected miRNAs and mRNAs, we conducted the transfection of OVCAR-3 and SKOV-3 cell lines by duplexes mimicking miR-203a, which was the miRNA with the greatest number of triplets. In the expression analysis, we considered the miR-203a-3p isoform because, according to miRBase, it is the most widespread mature sequence of miR-203a. The results of qRT-PCR analysis are shown in The expression level of CDK4, which does not have binding sites for miR-203, was very close to that of the mock control in both transfected cell lines. The level of WNT4, which also does not have binding sites for miR-203, was increased in OVCAR-3 and SKOV-3, apparently due to additional influences. A similar increase was observed for ZEB1 in OVCAR-3. The levels of c-MET and ZEB2  and a number of transcription factors, mainly the ZEB, SNAIL, and TWIST families, promote EMT ,27. As aIn the experimental part of this study, we analyzed expression levels of 13 mRNAs, 10 lncRNAs, and 5 miRNAs related to EMT in a representative set of primary ovarian tumors and matched normal tissues. All miRNAs were downregulated, meanwhile, the expression values for mRNAs and lncRNAs differed from those in in silico evaluation in available datasets. ZEB1, BCL2, and YAP1 among mRNAs and MLK7-AS1, LINC00339, and OIP5-AS1 among lncRNAs had a significant increase in expression level as expected. The decreased values and values with a null difference could be a result of the sample size and composition and/or data retrieval methods (Illumina sequencing or qPCR) and control samples (pooled norm or matched norm for each tumor sample) and require further analysis. Only two miRNAs formed triplets: miR-148a and miR-203a, and miR-203a had a number of connections with lncRNAs and mRNAs. Several studies demonstrated that miR-203a functioned as a tumor suppressor in ovarian cancer, whereas inhibiting this miRNA promoted tumor growth, migration, and invasion in a xenograft mouse model ,31. Morep < 10−4, FDR = 0.01). The strongest decrease in miR-203a expression in primary tumors from patients with metastases corresponded to a sharp appearance of hypermethylation of the MIR203A gene in primary tumors exclusively from patients with metastases. Here, we estimated all miRNA–mRNA, miRNA–lncRNA, and lncRNA–mRNA connections in which miR-203a was involved or associated . OVCAR-3 comprises epithelial cells isolated from the malignant ascites of a patient with progressive adenocarcinoma of the ovary (HTB-161). Notably, the OVCAR-3 cell line originates from metastases with ascitic fluid of a progressive adenocarcinoma. These differences between cell lines may explain the diverse effects of the transfection of miR-203a mimic on the mRNA of the studied genes. Nevertheless, the c-MET and ZEB2 levels were decreased in both OVCAR-3 and SKOV-3, which indicates the direct binding of miR-203a to these mRNAs.Once a malignant neoplasm has become metastatic, the tumor cells are found in the peripheral blood. Such circulating cells are the source of tumor RNA, which can be profiled to detect imbalanced triplets. Though the RNA concentration in plasma is significantly lower compared to that in tumor tissue samples, we suggest that the shift in the lncRNA–miRNA–mRNA interactome can still be determined.https://portal.gdc.cancer.gov/projects/TCGA-OV, accessed on 27 November 2021) for a tumor group and from the GTEx portal  for a normal group. The counts in datasets were normalized by FPKM (fragments per kilobase per million) values. Using the removeBatchEffect procedure from the limma package in R studio, we eliminated systematic errors that occur during the sequence alignment stage. The limma-voom function was applied for DE analysis of lncRNAs and mRNAs: only the expression variations across samples (log2IQR) > 0.5 with an adjusted p-value < 0.05 were considered significant. The false discovery rate (FDR) was controlled at the 0.01 threshold. We used TargetScan v.7.2, DIANA-TarBase v.8, and mirPath v.3 for sequence target prediction of the above-mentioned 12 miRNAs. The bioinformatic pipeline is presented in We extracted counts of reads from the TCGA-OV project, study accession phs000178 , with p-level of correlation were processed using the R studio cor.test function. For miRNA–lncRNA and miRNA–mRNA pairs, only negative correlations whose significance level was less than the specified threshold (p.adj < 0.05) were selected. The cutoff values of the correlation coefficient for the miRNA–lncRNA and miRNA–mRNA pairs were −0.33, while for lncRNA–mRNA, it was 0.4.We calculated Spearman’s correlation coefficients between expression levels for miRNA–lncRNA and miRNA–mRNA pairs. The target prediction for miRNAs was verified by TargetScan v.7.2, DIANA-TarBase v.8, and mirPath v.3. Then, the lncRNA and mRNA levels were analyzed to evaluate the regulatory relationships between these three RNA types in each triplet. The Spearman’s correlation coefficient and The study included paired samples of tumor and unchanged ovarian tissues from 46 patients, which were collected and morphologically characterized at the N.N. Blokhin National Medical Research Center of Oncology, Russia, Moscow. Most samples  were serous ovarian adenocarcinomas. Only the samples containing 70–80% or more tumor cells were used in the study. Patients did not receive chemotherapy prior to surgery. All samples were classified according to the TNM classification and histologically verified based on the WHO classification criteria. Clinical and morphological data are shown in 3 in volume obtained during biopsy or surgery were homogenized by an Ultra-Turrax T 10 basic disperser . Total RNA was isolated according to the guanidine–thiocyanate–phenol–chloroform extraction protocol with modifications as given previously [Samples up to 5 mmeviously . The totReverse transcription of RNA was performed using MMLV reverse transcriptase  and random hexanucleotide primers. Each setting of the reverse transcription reaction included negative controls containing no RNA.B2M gene was a reference gene.We estimated expression levels of 10 lncRNAs, 13 mRNAs, and 5 miRNAs by qPCR on a CFX96 thermal cycler . A SYBR Green intercalating dye, qPCRmix-HS SYBR (Evrogen), was added to the PCR mixture. The resulting mix was dispensed into a 96-well plate. The total volume of the PCR reaction was 20 μL. Conditions for amplification of DNA fragments were: 95 °C for 20 s—1 cycle; 95 °C for 1 s, 60–64 °C for 20 s—40 cycles. For lncRNAs and mRNAs, we used the primers presented in The expression levels of miRNAs were analyzed using TaqMan MicroRNA Assays : miR-124-3p (Assay ID: 001182), miR-148-3p (Assay ID: 00470), miR-191-5p (Assay ID: 002299), miR-203a (Assay ID: 000507), and miR-375 (Assay ID: 000564). RNU48 (Assay ID: 001006) and RNU6 (Assay ID: 001093) expression levels were used as references. The presence and length of the cDNA were checked by electrophoresis of PCR products in 1% agarose gel with an EtBr intercalating dye., and OVCAR-3 comprises epithelial cells isolated from the malignant ascites of a patient with progressive adenocarcinoma of the ovary (HTB-161).Two ovarian cell lines were used. SKOV-3 is a cell line with epithelial morphology that was isolated from the ovary of a 64-year-old White female with ovarian adenocarcinoma (HTB-77)v/v) fetal bovine serum (FBS) (Life Technologies), 50 µg/mL gentamicin (Life Technologies), 4 mM L-glutamine, and 4.5 g/L D-glucose, in a humidified atmosphere containing 5% CO2 in an incubator  at 37 °C. The cells were subcultured at confluence by treatment with 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS).Cell lines were purchased from the CLS Cell Lines Service GmbH . Cell line characteristics, including karyotype analysis data, were provided by the vendor. The identity of the cell line was not authenticated further. The cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% , resuspended at 200 μM, annealed by heating to 95 °C, and then slowly cooled to 37 °C. For transfection, 0.4 million cells were plated per well in a six-well plate supplied with 2 mL of medium (DMEM + 10% FBS). After 24 h of culture (at 50% to 80% confluence), the cells were supplied with fresh medium and transfected with an RNA duplex at a final concentration of 40 nM using Lipofectamine 2000 (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) following the manufacturer’s protocol. After 24 h, the cells were harvested and analyzed. Transfection of cel–miR-67-3p RNA duplexes exhibiting low homology with human miRNA sequences was used as a specificity control, whereas assessment of expression for hsa–miR-1-3p RNA duplexes together with their target twinfilin actin binding protein 1 (TWF1) was used as a positive control.https://cloud.genepattern.org, accessed on 23 March 2022).A Human Transcriptome Array 2.0 System (Thermo Fisher Scientific) was used for gene expression analysis. Biotinylated sense-strand DNA targets were prepared from 500 ng of total RNA using the GeneChip WT PLUS Reagent kit (Ambion). Hybridization, labeling, and washing were performed using the GeneChip Hybridization Wash and Stain Kit (Thermo Fisher Scientific) using Fluidics Station 450 (Affymetrix). The arrays were scanned using a Gene Scan 7G (Affymetrix) system. Standard Affymetrix quality control was conducted using the Expression Console Software (Affymetrix). Gene set enrichment analysis (GSEA) was applied for interpreting genome-wide expression profiles using GenePattern  was calculated using the same parameters as for in silico analysis. Differences were considered statistically significant at p.adj < 0.05.R studio was used to calculate median and interquartile range and to build the correlation matrix. The significance of differences was obtained by the nonparametric Mann–Whitney U test. The Spearman’s correlation coefficient (Highly expressed miRNAs have been suggested to be more significant for cancer diagnostics; however, poorly expressed miRNAs may also be markers for carcinogenesis. In the present study, we analyzed interactions of three types of RNAs according to the model of competitive endogenous RNA, in which miRNA is bound alternatively or competitively either to mRNA or lncRNA. In this case, miRNA acts as a mediator between both lncRNA and mRNA, while lncRNA and mRNA may compete with each other for miRNA binding. We showed for the first time that the downregulated tumor-suppressive miR-148a and miR-203a induced an imbalance in their RNA interactome. In experimental validation, we focused on EMT-related lncRNAs and mRNAs. Two triplets attracted attention, OIP5-AS1–miR-203a–ZEB2 and OIP5-AS1–miR-203a–c-MET, due to the strong correlation between expression levels of lncRNA, miRNA, and mRNA players and due to the presence of miR-203a binding sites in OIP5-AS1, ZEB2, and c-MET. The interaction of miR-203a with ZEB2 was revealed for the first time in OC, and with c-MET for the first time in oncogenesis. The possibility of miR-203a direct binding to ZEB2 and c-MET was shown by transfection of the OVCAR-3 and SKOV-3 cell lines with the miR-203a mimic. The high tissue specificity of such interactions benefits the expression profiling for diagnostics and prediction of a number of cancers, including OC.


For determining the immobilization steps and optimization of the biosensor, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were used. Furthermore, the morphological characterization of each ITO electrode surface was performed by field emission scanning electron microscopy (FESEM). Specific binding of gold nanoparticles supported CTAB to ITO-PET and its bioconjugation with the biotinylated antibody of HA was studied using the electroanalysis of the sensor performance. For the better performance of the antibody to generate an immunocomplex with HA (antigen), its encapsulation was performed, which led to the excellent behavior of the immunosensor. The proposed HA immunosensor indicated excellent reproducibility, high selectivity, and long-term stability. The HA electrochemical immunosensor performed perfectly with a wide determination range (0.078 to 160 ng mL−1) and a low limit of quantification (0.078 ng mL−1) in human plasma samples. It is recommended that the designed biosensor can be used as a diagnostic tool in clinical bioassays in the near future.Hyaluronic Acid (HA) is a non-sulfated glycosaminoglycan, which is a potential biomarker that could be evaluated in the diagnosis of some cancers. For the first time, a novel label-free electrochemical immunosensor was developed based on modified ITO-PET  electrodes for the sensitive recognition of hyaluronic acid (HA) in real samples. A disposable ITO-coated PET electrode was modified with gold nanoparticles (AuNPs) to construct a suitable substrate for the efficient immobilization of biotinylated antibodies of HA. Importantly, the encapsulation of biotinylated antibody of HA in KCC1-NH-CS Hyaluronic Acid (HA) is a non-sulfated glycosaminoglycan, which is a potential biomarker that could be evaluated in the diagnosis of some cancers. Hyaluronic acid is also the only GAG that does not bind to core proteins and undergo post-synthetic modifications.6 Long hyaluronic acid polymers have the ability to bind to large amounts of water. The hygroscopic and viscoelastic properties of HA make it a perfect component of the vitreous humor, synovial fluid and dermis. Hyaluronic acid in its very long polymer native form is known as high molecular weight (HMW) hyaluronic acid.7 However, under certain conditions, it can be broken down into smaller fragments called low molecular weight HA (LMWHA). Hyaluronic acid turnover is a rapid process as the half-life of HA molecules in the bloodstream is only 2–5 minutes.7 HA levels are significantly related to joint diseases. HA is the primary ligand for CD44 that is overexpressed in many cancer types including pancreatic, breast, lung, ovarian, and prostate.8 HA promotes tumor growth, metastasis and angiogenesis, which have shown the potential to be diagnostic and/or prognostic biomarkers in a few malignancies.9 HA family biomarkers promote bladder cancer (BCa) growth and progression very likely by upregulating the epithelial–mesenchymal transition (EMT) phenotype.9 Elevated HA in urine is an accurate diagnostic method for the efficient detection of BCa, regardless of tumor grade and stage.10 Recently, a research group determined HA by enzyme-linked immunosorbent assay11 and they showed the presence of this potential biomarker in the urine of high-risk patients for prostate cancer. Thus, HA showed a significant predictive ability for prostate cancer. With an optimal cut-off point of 50.13, HA has a 65% sensitivity and 53.9% specificity.11 Patients with HA levels more than 50.13 ng mL−1 had a 2.31 times greater likelihood of prostate cancer.11 A high level of HA can predict unfavorable breast cancer outcomes.12 Also, a high plasma level of HA is significantly associated with poor prognosis of metastatic breast cancer (MBC) patients. HA plasma levels decreased in parallel with the treatment response, signifying that HA could also be an effective biomarker for MBC treatment monitoring.13 HA accumulation has been found in different human digestive cancers and predicts advanced tumor stages, early recurrence, and poor prognosis of the patients. HA may be used to identify potentially high-risk groups of upper gastrointestinal cancers including gastric noncardia cancer and esophageal squamous cell carcinoma, particularly in high incidence areas.14 A high percentage of pericellular HA-positive cancer cells and a high intensity of the stromal HA signal correlate with lower cancer-related survival rates and recurrence-free rates among colorectal cancer (CRC) patients.15 However, there is a limited information about the prognostic value of serum HA levels in hepatocellular cancer (HCC) patients.15 Detection of special and sensitive biomarkers for every cancer is an urgent need, which could help postpone initiation of symptoms with early intervention, decreasing mortality rates, particularly for early diagnosis. HA could be a new prognostic biomarker useful for the selecting individual therapeutic strategies for cancer patients, therefore, early and sensitive detection of this marker is important for clinicians.Hyaluronic acid (HA) is an unbranched glycosaminoglycan consisting of the repeating disaccharide units of 16 plasmonic mass spectrometry,17 colorimetric, and immuno-electrophoresis.18,19 However, these methods have some limitations, such as requiring a time-consuming process, having laborious sample preparation steps, unsuitable for reusability, and being more expensive. Biosensors measure target molecules within a certain detection interval in a biological sample and are analytical measurement devices that transform this information into a meaningful electrical signal.20 Improving the performance of biosensors, particularly in clinical applications, is still urgently requested in this field. The development of biosensors has basically allowed simple, low-cost diagnostics and disease screening efficiently.21 Biosensors are being focused on developing highly sensitive and specific biomarker recognition to detect the disease at an early stage.22 Recently, a simple but sensitive electrochemiluminescence (ECL) biosensor has been designed for HAase detection based on the electrostatic interaction of anionic hyaluronic acid (HA) and cationic luminous reagent Ru(bpy)32+. The proposed ECL system has been applied to detect HAase in urine samples with satisfying results and HAase inhibitors with high efficiency.23 A room-temperature phosphorescent (RTP) PDAD–Mn–ZnS QD biosensor was fabricated using poly  (PDAD) as the modifier of MPA-capped Mn–ZnS QDs, and was used for HA determination. This proposed RTP sensor can avoid interference from matrix background fluorescence or stray light that occurs during fluorescence measurements. Therefore, this biosensor is potentially suitable for detecting HA in actual samples without complicated pretreatment.24–28The increasing requirements for fast and reliable sensing devices stimulate the development of novel technological solutions, the conventional methods used for the bioassay of HA mainly include enzyme-linked immunosorbent assay (ELISA), a competitive fluorescence-based assay,28 Additionally, a suitable surface, a wide working range, material stability, and linkage strength are their unique advantages.29 Also, the ITO-PET electrode is very cost-effective compared to other conventional working electrodes  In this study, we used a novel immunosensor based on ITO PET disposable electrode for the identification of HA using an electrochemical method.One of the most popular conductive oxides is ITO-based electrodes. Features that add to the popularity of this type of electrode are their transparency and excellent electrical properties.25 Using an ITO film, electrodes for biosensors have been capably increased because of their features, such as high electrical conductivity, low electrochemical background response, low cost, simple preparation for sensing applications, wide working area, corrosion resistance, and reusability.26 Until now, numerous biosensors have been developed by using ITO as a substrate for the working electrode, such as electrochemical nucleic acid biosensors, immunosensor, and microfluidic on-chip sensors.25 In this study, gold nanoparticles were chosen as the conductive material to modify an ITO electrode in order to enhance the electrical characteristics of the ITO substrate. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were used to investigate the electrochemical properties of the modified electrodes at each step of the biosensor fabrication and survey the formation of Ab–Ag immunocomplex, which is necessary for the recognition of HA in real samples. The field emission scanning electron microscopy (FESEM) technique was used for the characterization of the surface morphology of the biosensors as well. Reproducibility and repeatability and regeneration parameters were also examined for the determination of analytical characterization of the fabricated biosensor. To expound the binding characterization of HA and anti-HA, a single frequency impedance method was used as well.We have developed a novel label-free electrochemical immunosensor based on modified ITO-PET  electrodes for early detection of HA in real samples. ITO-based biosensors are utilized in many fields such as clinical diagnosis, and environmental monitoring.2.2.1.−2) were purchased from NanoGostar Sepahan . Hyaluronic acid kit containing Biotin-HA-Ab and 320 ng mL−1 main concentration of HA-antigen was acquired from ZellBio GmbH (Germany) by Padgin Teb Company. Hydrogen tetrachloroaurate (iii) hydrate (HAuCl4–3H2O), cetyltrimethylammonium bromide (CTAB), and sulfuric acid, 98%, were obtained from ''Sigma-Aldrich '', for performing electrochemical deposition of Au nanoparticles on ITO-PET electrodes. An electrochemical investigation of the detection zone of the ITO-PETs was implemented using a ferricyanide/ferrocyanide solution including 0.5 M of K4Fe CN6/K3Fe CN6 along with KCl. Fresh frozen plasma samples were obtained from the Iranian Blood Transfusion Research Center . KCC-1-NH-CS2 was used for encapsulation and activation of biotinylated antibody that was synthesized by our research group, previously.27Five indium tin oxide coated polyethylene terephthalate film (ITO-PET) electrodes , SWV with the current ranges of 10 nA–100 μA  and CV with the current ranges of 1 nA–100 μA  techniques were applied for the primary detection of HA. The resistance investigation of each modified electrode was performed using the EIS technique with the current ranges of 100 pA–1 mA . The chronoamperometry technique  was used for the electrochemical deposition of Au nanoparticles on ITO-PET electrodes. The surface morphology of the detection zone was recognized using high-resolution field-emission scanning electron microscopy  with a working voltage of 3–6 kV, and the chemical compounds of the electrodes were examined using energy-dispersive spectroscopy (EDS) along with FE-SEM.For electrochemical measurements, a three-electrode cell (from Metrohm), involving a Pt wire as a counter electrode, Ag/AgCl-saturated KCl as a reference electrode and ITO-PET as the working electrode was used, which was powered by an electrochemical system connecting the PalmSens system with PS4.F1.05 . The system was run on a PC using PSTrance 5.3 software. In this investigation, differential Pulse voltammetry with the current range of 100 pA–100 μA  In this study, we used a novel immunosensor based on the ITO-PET disposable electrode for the identification of HA using an electrochemical method. The first and most essential condition in the electrochemical experiment that affects the recognition process, is the modality and transparency of the active surface of ITO-PET electrodes. Contaminated environments and surfaces in such experiments affect the electrochemical reactions and as a result, cause incorrect and unwanted outcomes. Thus, a cleaned and prim surface is important before modification and bioanalysis. For this purpose, prior to the modification, the ITO surface was cleaned by sonication in acetone and distilled water for 10 minutes, then dried using nitrogen gas for 30 seconds.One of the most popular conductive oxides is the ITO-based electrode. Features that add to the popularity of this type of electrode are their transparency and excellent electrical properties.2.4.Tequilibration = 2 s, Edc = −0.23 V, tinterval = 0.1 s, trun = 120 s. The deposition was performed on the uninsulated surface .4–3H2O'' solution was prepared, then, CTAB 10% was dissolved in sulfuric acid at a concentration of 200 mM. After all, 10 mM of the “HAuCl4–3H2O” solution was added (With a ratio of 1 : 1 : 1) to the previously prepared solution. The container was shaken gently to obtain a homogeneous solution. The color of the solution was changed to orange. 10% CTAB solution was used as a pattern and capping agent to form a regular shape and protect the nanoparticles from agglomeration, also a sulfuric acid solution was used as a solvent solution.Au nanoparticles were synthesized using CTAB and sulfuric acid. First, 10 mM ''HAuCl2.5.2 were synthesized following the technique described in our prior research.30 Dithiocarbamate-functionalized KCC-1-NH2 was prepared as follows: 100 mg of CS2 was dissolved in 20 mL of acetonitrile containing 1 percent Et3N and 90 mg of KCC-1-NH2. The resulting mixture was swirled overnight, to ensure complete dissolution. Finally, an evaporator was used to dry the solution  were employed to encapsulate the HA antibody. As a result, biotinylated Ab, which was immobilized on the surface of the ITO-based electrodes, was trapped in KCC-1-NH-CS2. For this purpose, 0.001 g KCC-1-NH-CS2 was first dissolved in the phosphate buffer (pH 7.4). Then, 300 μL of biotinylated antibody was added to the above-mentioned solution and gently stirred for 24 hours at room temperature on a shaker. Therefore, after centrifuging the mixture at 6000 rpm for 5 minutes  and incubated for 3 hours at a temperature of 22 °C because the nanocomposite is sensitive to temperatures above 22 °C. Phosphate buffer (with pH 7.4) was used for preparing the KCC-1-NH-CS2 solution. In order to encapsulate and activate biotinylated Ab, 100 μL of phosphate buffer was combined with 0.001 g of KCC-1-NH-CS2 and added to 300 μL of antibody, placed on a shaker for 24 hours at room temperature. Then, centrifuged for 5 minutes at 6000 rpm and the resulting supernatant was used for investigations. After incubation of Ab, 5 μL of BSA protein solution (10%) was drop-casted on the modified detection zone (ITO-PET-Au-Ab) as a blocking agent and incubated for 40 minutes. Finally, 5 μL of HA antigen was immobilized on ITO-PET-Au-Ab-BSA, this step lasted for 2 hours. After washing the modified surface with DW, it was translocated into an electrochemical cell containing K4Fe(CN)6/K3Fe (CN)6/KCl 0.5 M for electroanalysis. After depositing Au on ITO-PET electrodes, it took 20 minutes for the surface to dry completely. Then, 5 μL of HA Biotin-Ab that was encapsulated and activated with KCC-1-NH-CS3.1.4Fe(CN)6/K3Fe (CN)6/KCl 0.5 M . The addition of Ab seems to play an important role in its formation. Evaluation of ITO-PET-Au(CTAB)-Ab-BSA  were selected for the analysis of the morphology of the nanocomposite. As shown in )-Ab-BSA  was the )-Ab-BSA . At this2 and efficiently immobilized on the surface of ITO-PET modified AuNPs(CTAB). The addition of Ab seems to play an important role in its formation. Evaluation of ITO-PET-Au(CTAB) -Ab-BSA was the third step in imaging.As shown in Fig.The arrangement of the nanocomposite at this stage is almost the same as the previous stage so the nanoparticles are compactly placed next to each other. The last imaging step was the evaluation of ITO-PET-AuNPs(CTAB) -Ab-BSA-Ag.3.3.EDS and MAP analysis were applied in this work towards elementary discovery. Atomic-resolution chemical mapping using energy-dispersive X-ray spectroscopy of different steps of the fabricated immunosensor revealed valuable data. The analysis of carbon, oxygen, potassium, phosphor, nitrogen, and gold elements was investigated. A–C figures are related to ITO-PET bare electrode . As can 4.4.1.In this research, DPV, SWV, and EIS were applied for the bioanalytical study of the immunosensor toward the measurement of HA. This trend is important and predictable, as the current intensity decreases with a decrease in the concentration and subsequent changes in peak height, as seen in the DPV and SWV plots . In othe4.2.−1). There is also a clear linear relationship between different concentrations. The linear relationship can be expressed by the recoded regression equation in the figures. The results (−1). There is also a clear linear relationship between different concentrations. The linear relationship can be expressed by the recoded regression equation in the figures. Comparing the graphs obtained in the previous step with the graphs of a real sample shows that the current intensity is slightly higher in relation to a real sample, and this difference can be related to the nature of the sample  and bioreceptor degradation over time.2 see ESI, the sta2 see ESI. The res2 see ESI) show th4.4.39 Ideal reproducibility is one of the most important features of the system designed in this study. As can be seen in the results  for the SWV technique and SD = 1.21 (n = 3) for the CV technique were recorded. Also, to confirm the proper reproducibility of the system developed in this study, the repeatability of the biosensor was evaluated using the CV technique. The results , the bi4 see ESI, SD = 1.5 see ESI obtained4.5.40 The results 6/K3Fe(CN)6. The results of CV as a function of the scan rate of the AuNPs-CTAB electrodes are shown 6/K3Fe(CN)6 redox couple. The anode and cathode peak currents were found to increase linearly at different potential sweep rates in the range of 10 to 1000 mV s−1, confirming the diffusion-limiting behavior of the redox couple electrochemical reactions. It can be concluded that a slow sampling rate slows electron exchange on the electrode surface, records peak currents, shortens peak currents, and narrows the voltammogram. From the linear dependence of the peak current on the square root of the sampling rate and the Napier logarithm of the peak current on the Napier logarithm of the sampling rate. We conclude that the reaction is diffusion controlled.Cyclic voltammetry was used to evaluate the electrode-catalytic behavior of AuNPs-CTAB electrodes and the redox behavior of K (see ESI)). The a5.2 was reported for the first time in this study. In this work, KCC-1-NH-CS2 acts as a green, efficient, and reusable nanomaterial. In addition, the developed HA-based immunosensor has suitable LLOQ “(0.078 ng mL)” and has a wide linear detection range “(0.078 to 160 ng mL−1)”. In summary, we developed an HA-based biosensor that is regenerative, sensitive, practical, low-cost, and has a wide detection range. It has considerable potential in cancer biomarker diagnosis.In this work, a label-free electrochemical immunosensor for the sensitive and specific detection of HA was designed by using transparent ITO-PET electrodes. The resulting immunosensor has various advantages, including high sensitivity, good selectivity, long storage stability, excellent reproducibility, and simplicity to fabricate and function. Antibody encapsulation inside KCC-1-NH-CSHAHyaluronic acidITO-PETIndium tin oxide-polyethylene terephthalateAuNPsGold nanoparticlesEISElectrochemical impedance spectroscopyCVCyclic voltammetryFESEMField emission scanning electron microscopyCTABCetyltrimethylammonium bromideCDCluster of differentiationHAasesHyaluronidasesECMExtracellular matrixGAGsGlycosaminoglycansHMWHigh molecular weightLMWHALow molecular weight HABCaBladder cancerEMTEpithelial–mesenchymal transitionMBCMetastatic breast cancerCRCColorectal cancerHCCHepatocellular cancerELISAEnzyme-linked immunosorbent assayECLElectrochemiluminescenceRTPRoom-temperature phosphorescentPDADPoly (diallyldimethylammonium chlorideCA15-3Cancer antigen 15-3Data sharing is not applicable. It will be shared if needed.Conceptualization: Sanam Dolati and Mohammad Hasanzadeh data curation: Fereshteh Kohansal formal analysis: Fereshteh Kohansal funding acquisition: Sanam Dolati investigation: Ahmad Mobed methodology: Mohammad Hasanzadeh project administration: Sanam Dolati Software: Fereshteh Kohansal supervision: Sanam Dolati and Mohammad Hasanzadeh validation: Mohammad Hasanzadeh, Writing–original draft: Ahmad Mobed and Sanam Dolati, writing –review and editing: Mohammad Hasanzadeh.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.RA-012-D2RA04984H-s001RA-012-D2RA04984H-s002RA-012-D2RA04984H-s003RA-012-D2RA04984H-s004RA-012-D2RA04984H-s005RA-012-D2RA04984H-s006RA-012-D2RA04984H-s007RA-012-D2RA04984H-s008RA-012-D2RA04984H-s009RA-012-D2RA04984H-s010RA-012-D2RA04984H-s011


Background: Limited graft availability is a constant clinical concern. Hence, the umbilical cord (UC) is an attractive alternative to autologous grafts. The UC is an inexhaustible tissue source, and its removal is harmless and part of standard of care after the birth of the baby. Minimal information exists regarding the immunological profile of a whole UC when it is considered to be used as a tissue graft. We aimed to characterize the localization and levels of class I human leukocyte antigens (HLAs) to understand the allogenicity of the UC. Additionally, HLA-E and HLA-G are putative immunosuppressive antigens that are abundant in placenta, but their profiles in UC whole tissue are unclear. Hypothesis: The UC as a whole expresses a relatively low but ubiquitous level of HLA-ABC and significant levels of HLA-G and HLA-E. Methods: Healthy patients with no known pregnancy-related complications were approached for informed consent. UCs at term and between 12 and 19 weeks were collected to compare HLA profiles by gestational age. Formalin-fixed paraffin-embedded tissues were sectioned to 5 µm and immunohistochemically stained with a pan-HLA-ABC, two HLA-G-specific, or an HLA-E-specific antibody. Results: HLA-ABC was consistently found present in UCs. HLA-ABC was most concentrated in the UC vessel walls and amniotic epithelium but more dispersed in the Wharton’s Jelly. HLA-E had a similar localization pattern to HLA-ABC in whole UC tissues at both gestational ages, but its protein level was lower. HLA-G localization and intensity were poor in all UC tissues analyzed, but additional analyses by Western immunoblot and mass spectrometry revealed a low level of HLA-G in the UC. Conclusion: The UC may address limitations of graft availability. Rather than the presence of HLA-G, the immunosuppressive properties of the UC are more likely due to the abundance of HLA-E and the interaction known to occur between HLA-E and HLA–ABC. The co-localization of HLA-E and HLA-ABC suggests that HLA-E is likely presenting HLA-ABC leader peptides to immune cells, which is known to have a primarily inhibitory effect. One of the constant disadvantages of clinically using autologous tissue grafts is limited graft availability . One exaThe UC is an extraembryonic tissue that serves as a bidirectional transport system between the fetus and the placenta to support fetal development during pregnancy . It is cThe immunosuppressive properties of CLECs and UCMSCs have mainly been attributed to the presence of the non-classical MHC-I molecule HLA-G ,24,27,28Despite growing interests in using the UC as tissue grafts, little evidence exists regarding the localization and levels of class-I HLA molecules in the context of the whole cord. Here, we investigate and compare the localization and levels of HLA-ABC, HLA-G, and HLA-E in whole-cord cross-sections to better understand the allogenicity of the UC.n = 10, n = 10, 12–19 weeks). For samples collected at term, the decidua and trophoblast villi were dissected as whole tissue blocks from the placenta, and the entire length of the UC was removed at 2 cm from its maternal end immediately following delivery. Decidual tissues and UC segments from early pregnancy were rinsed and collected in phosphate buffered saline (PBS). Tissue samples were gently washed in PBS to remove residual blood and submerged in PBS at 4 °C until fixation. Tissue samples were also dissected to small fragments, flash frozen in liquid nitrogen, and stored at −80 °C.This study was approved by the Sunnybrook Health Sciences Centre Research Ethics Board (#136-2008), and all patients provided informed consent. Term placental and UC samples n = 10,  were colImmunohistochemistry (IHC) staining against HLA-ABC, HLA-G, and HLA-E were performed on UC tissue cross-sections that were collected from term pregnancies and between 12 and 19 weeks. Gestational-age-matched placental samples were used during the initial IHC protocol optimization process and as positive controls for all subsequent experiments. A mouse IgG1 antibody isotype  was used as a negative control at the same working concentration as the corresponding primary antibodies. Tissues were dissected and fixed in 10% neutral buffered formalin (NBF) within 24 h of collection. To determine the optimal fixation conditions for the UC, UC samples were dissected to approximately 5 mm thick cross-sections and fixed for 24 or 48 h, with or without rotary agitation, and at 4 °C or room temperature. Upon comparing the HLA-ABC staining results of UC tissue cross-sections at term, the optimal fixation conditions were determined to be 48 h with rotary agitation at room temperature. Placental samples were fixed at 4 °C overnight with gentle rotary agitation, washed twice in PBS for 20 min each, post-fixed in 10% NBF for 30 min, and washed twice in PBS for 20 min, each at room temperature. All fixed tissue samples were submerged in 70% ethanol at 4 °C until paraffin embedding.2O), and washed twice in PBS for 5 min each. Five different antigen retrieval (AR) protocols were tested on placental and UC samples to optimize the tissue preservation and epitope exposure for each tissue type and antibody. Two heat-induced epitope retrieval (HIER) protocols were tested: microwave and pressure cooker (PC). With the microwave protocol, slides were placed in a slide container and completely submerged in 0.01 M sodium citrate buffer (pH 6). Then, the submerged slides were heated in the microwave at power six for 5 min, cooled for 20 min at room temperature, heated again at power six for 4 min, and cooled again for 20 min at room temperature. If there were vacant slots in the slide container, blank slides were used to ensure all slots were filled. Doing this helped facilitate a uniform distribution of the heat to each slide, which would in turn reduce differences in the staining intensity and tissue morphology due to uneven heating. With the PC protocol, slides were laid flat inside a microwavable PC and submerged in 0.01 M sodium citrate buffer (pH 6) and heated using a microwave at power 10 for 8 min. Slides were then cooled at room temperature for 45 min. Two enzymatic AR protocols were tested: 0.25% trypsin (1.1 mM in PBS) (Corning) and proteinase K (20 µg/mL in PBS) . Tissue sections were encircled with an ImmunoPen  and incubated with proteinase K or trypsin for 10 min at 37 °C. In the fifth condition, no AR was performed. Upon optimization, HIER with the microwave was used for all placental samples and for staining HLA-E in UC samples. Enzymatic AR with proteinase K was used for staining HLA-ABC and HLA-G in UC samples.Formalin-fixed paraffin-embedded tissue samples were sectioned to 5 µm on Superfrost Plus slides (Electron Microscopy Sciences), with each slide containing two serial sections of one sample. Slides were baked at 55 °C for 1 h prior to IHC staining to enhance tissue adhesion to slides. Slides were deparaffinized in xylene, rehydrated in a decreasing gradient of ethanol in distilled water (dHv/v) (TBS-T) for 1 h at room temperature. Without washing, one tissue section of each slide was incubated with the primary antibody, and the other was incubated with the mouse IgG1 antibody as the negative isotype control  at 4 °C overnight. The primary antibodies used were a mouse monoclonal anti-human pan HLA-ABC antibody at 0.25 µg/mL , two different mouse monoclonal anti-human HLA-G antibodies at 2.0 µg/mL  , and a mouse monoclonal anti-human HLA-E at 5.0 µg/mL . The negative isotype controls were used at the same working concentration as corresponding primary antibodies. After washing the slides twice in PBS for 5 min each, slides were incubated with a goat anti-mouse biotin secondary antibody at 2.6 µg/mL  for 1 h at room temperature. For both primary and secondary antibodies, 5% normal goat serum in TBS-T was used as the diluent. After two 5 min PBS washes, slides were incubated with the VECTASTAIN Elite Avidin Biotin Complex Peroxidase Kit  for 30 min and developed with a 3,3′-diaminobenzidine substrate kit  for 5 min at room temperature. Slides were washed once in dH2O for 3 min, counterstained with the Gill No. 2 Hematoxylin (Millipore Sigma) for 30 s, and washed in warm running tap water for 5 min. Finally, slides were dehydrated in 70%, 90%, and 100% ethanol, cleared in xylene, mounted with Cytoseal 60 , and imaged with the TissueScope LE Slide Scanner . Staining results were graded based on staining intensity as follows: 0 (no staining or distinct differences from the isotype control), + (weak brown staining), ++ (moderate brown staining), +++ (strong dark brown staining).Following AR, slides were washed twice in PBS for 5 min each and incubated in 3% hydrogen peroxide in methanol for 30 min at room temperature to quench the endogenous peroxidase activity in the tissue. Slides were washed twice in PBS for 5 min each, and non-specific binding was blocked by incubating slides in 5% normal goat serum  in Tris-buffered saline with 0.1% Tween 20   for 6 min. Following one wash in tap water for 2 min, the slides were de-stained with 1% acid alcohol (Leica) for 6 s. Then, slides were washed in tap water for 4 min and dipped in 80% ethanol for 30 s. The slides were counterstained with eosin (Leica) for 15 s and were then dehydrated by being dipped once in 95% ethanol for 10 s and twice in 100% ethanol for 30 s each. Finally, slides were cleared in xylene and mounted with Cytoseal-XYL (Richard-Allan Scientific). Slides were imaged with the TissueScope LE Slide Scanner (Huron).Hematoxylin and eosin (H&E) stains of UC and placental tissue sections were performed. Tissue sections were deparaffinized in xylene and rehydrated in a decreasing gradient of ethanol in dHg for 5 min at 4 °C to collect the tissue lysate supernatant. Cell lysates were collected as previously described [g for 5 min at 4 °C to remove insoluble cell debris.The lysis buffer used for both tissue and cell lysates was radioimmunoprecipitation assay (RIPA) buffer (NaCl (150 mM), Nonidet P-40 (1%), sodium deoxycholate (0.5%), SDS (0.1%), and Tris ) containing cOmplete ULTRA protease inhibitor cocktail . Tissues stored at −80 °C were thawed and grinded in RIPA buffer on ice until they were mostly homogenized. Then, tissues were allowed to further digest in solution on ice for 1 h and centrifuged at 1000× escribed . BrieflyWestern blot analysis was performed as previously described using equal amounts of total protein from UC tissue lysates, with placental tissue lysates as positive controls and primary human microvascular endothelial cell (HMVEC) lysates as negative controls . All wasw/v) for 1 h. Following three washes of TBS-T for 5 min each, membranes were incubated with a mouse monoclonal anti-human HLA-G antibody  diluted to 1:500 (v/v) with TBS-T at 4 °C overnight. After three washes of TBS-T for 5 min each, membranes were incubated with a goat anti-mouse IgG secondary antibody  diluted to 1:2000 (v/v) with 5% nonfat dry milk in TBST (w/v) for 1 h. The protein actin was used as the housekeeping protein and was probed on the same membranes that were used to detect HLA-G presence. The protocol used to probe for actin was the same as the one described for HLA-G unless otherwise specified. After the membrane was striped, non-specific binding was blocked again before primary antibody incubation. The primary antibody used was a polyclonal goat anti-actin antibody  diluted to 1:2000 (v/v) with TBS-T, and the secondary antibody used was a polyclonal donkey anti-goat IgG antibody . Finally, all immunoblots were visualized by enhanced chemiluminescence substrate Western Lightning Plus-ECL  and imaged using the Chemidoc Imaging System .Briefly, 25 µg of total protein from lysates was added to the loading sample buffer with 5% β-mercaptoethanol (βME) (Millipore Sigma), heated at 95 °C for 5 min, then electrophoresed on a 10% SDS-PAGE gel. Then, proteins were transferred to polyvinylidene difluoride membranes , which were briefly soaked in methanol for 30 s and acclimated in TBS-T prior to protein transfer. Then, membranes were washed three times in TBS-T for 5 min each and non-specific binding was blocked by incubating membranes in 5% nonfat dry milk in TBS-T (Immunoprecipitation was performed to purify HLA-G molecules from UC tissue lysates prior to mass spectrometry (MS) analysis. All washes and incubation periods were performed on the rotator at 4 °C unless otherwise specified. For each lysate mixture, 20 µL of magnetic beads  were aliquoted and washed three times with PBS for 3 min each. To attach the mouse monoclonal anti-human HLA-G antibody  to the beads, the antibody was diluted to 1:100 in PBS with 0.02% Tween 20 and incubated with the beads overnight. After removing the supernatant with a magnetic rack, 1 mg of total protein from UC tissue lysates was added to the antibody-attached beads and incubated overnight to purify HLA-G molecules from lysates. HLA-G molecules in the tissue lysates were now attached to the anti-HLA-G antibody on the magnetic beads. After removing the supernatant, the beads mixtures were first washed three times in RIPA buffer containing the cOmplete ULTRA protease inhibitor cocktail (Roche Diagnostics) for 10 min each, and they were then washed three times in Tris-EDTA buffer  for 10 min each. After removing the buffer, 20 µL of the sample buffer with 10% βME was added to each beads mixture and heated at 95 °C for 10 min to elute the HLA-G-antibody complex from the beads. Then, the mixtures were briefly vortexed and centrifuged to collect the supernatant, which contained the purified HLA-G. They were then stored at −80 °C.2O in a clean tray, then incubated in the Coomassie blue stain (Abcam) overnight with gentle agitation at room temperature. To wash out excess Coomassie blue, the gel was washed for 4 h in dH2O with a change of clean dH2O every hour. Finally, the band of interest was excised from the gel and analyzed by MS with a blank area of the gel as negative control. The resulting data were interpreted using the Biopharma Finder software (Thermo Fisher).To prepare samples for the MS analysis, the denatured and purified protein samples were electrophoresed on a 10% SDS-PAGE gel. The gel was briefly washed with dHSince HLA-ABC is expressed in almost all nucleated cells in adults, it is expected to be also present in the placenta and UC. Therefore, the IHC protocol optimization of placental and UC samples was conducted based on the HLA-ABC staining results. The criteria used to select the optimal IHC protocol using HLA-ABC staining results included minimal physical damage to tissue sections during staining, a consistent staining intensity, and minimal non-specific binding.To begin the optimization process, three different working concentrations of the anti-HLA-ABC antibody  and five different AR protocols were tested using placental tissue sections . The AR To highlight the morphological differences within the WJ and offer further clarity when describing the IHC staining results of UC tissue cross-sections, we will refer to specific parts of the UC by the anatomical terminologies defined in a review from 2017 . The amnThe optimal fixation conditions and AR protocol were determined for UC samples using UC tissue sections at term . Due to The HLA-ABC IHC staining results of all tested AR protocols in UC tissue cross-sections at term can be found in Across all UC regions, group 2 had the strongest staining intensity . The staThe IHC staining results of HLA-ABC, HLA-G, and HLA-E in UC tissue cross-sections were scored based on their staining intensities and irrespective of the prevalence of positive staining. The localization of HLA-ABC and HLA-E at the AE were similar among UC tissues at term and between 12and 19 weeks , but theThe positive staining of HLA-ABC and HLA-E was noted specifically in cells of the WJ subregions at both gestational ages, though not all WJ cells showed positive staining . HLA-ABCHLA-ABC and HLA-E had similar localization patterns in the umbilical vessels of UC tissues at term. A moderate to strong staining intensity was observed in the endothelial cells after staining for either HLA-ABC or HLA-E . CompareOverall, for UC tissues between 12 and 19 weeks, HLA-ABC levels were higher than HLA-E at both the endothelium and vessel walls . At the A final comparison was made on the localization and levels of HLA-ABC and HLA-E between UCs at term and at 12–19 weeks. HLA-ABC and -E had similar localizations in most UC samples analyzed, regardless of gestational age . HLA-ABCAdditional experiments were performed to further investigate the unexpected lack of positive HLA-G IHC staining in all UC tissues analyzed. Western immunoblotting was performed on whole UC tissue lysates to determine HLA-G presence in the UC . The domTo further confirm the presence of HLA-G in UC, we analyzed the 55 kDa dominant band in whole UC tissue at term using MS. HLA-G molecules from the UC tissue lysate was affinity purified by immunoprecipitation, and the 55 kDa band was excised from the Coomassie blue-stained gel. From the same gel, a blank area was excised and used as a negative control. The MS analysis revealed the presence of the HLA-G isoform G5 in the 55 kDa band . SpecifiWe sought to determine the localization and abundance of various class I HLA molecules to understand the immunogenic and immunosuppressive profiles of the UC. We observed HLA-ABC to be present throughout the UC, with its highest abundance in the AE and endothelium. We observed minor levels of the immunomodulatory HLA-E within the UC, and most of it was present in the AE, WJ, and the endothelium. HLA-E was not consistently present in all patient UC samples. The IHC staining of the UC consistently showed an absence of HLA-G regardless of gestational age, but further investigation by Western immunoblot and MS analyses revealed a presence of the HLA-G5 isoform at low abundance in UC tissues at term.To our knowledge, this study is the first to understand the relative localization and levels of these important immune-regulatory molecules in whole UC tissue. Upon initial examination, UCs from first-trimester patients revealed very poor structural integrity. The physical characteristics of first-trimester UCs steered our efforts towards second- and third-trimester UCs since future applications would require UC lumens to be physically manipulatable. Overall, we observed higher levels of HLA-E staining intensity in UC tissues at term compared with those in the second trimester, while HLA-ABC levels were similar in both gestational ages. Regardless of gestational age, we did not observe any positive HLA-G staining using IHC in any of the UC tissues.Based on our findings, discordance exists regarding the presence of HLA-G in the UC. Two anti-HLA-G antibody clones were used in the IHC staining of UC tissues: 4H84 and MEM-G/1. HLA-G5 was detected to be present in the UC tissue by Western immunoblot and MS analyses, but no positive HLA-G staining was observed by IHC. In contrast, both 4H84 and MEM-G/1 consistently detected HLA-G presence in placental tissues in early pregnancy and at term, which were used as positive controls. This shows that the reliability and sensitivity of 4H84 and MEM-G/1 are sufficient for HLA-G detection in placental tissues but not in UC tissues where the HLA-G level is likely low as we observed. A similar lack of concordance was noted in the detection of HLA-G in rectal cancer tissue . SpecifiBased on the observations reported in the current study, HLA-G may not be the main contributor of the immunosuppressive properties observed in UC allografts. Furthermore, a recent study reported an absence of HLA-G expression and high levels of HLA-ABC expression in UCMSCs using 10x single-cell RNA sequencing analysis . Though + cells are protected from NK cell lysis [To our knowledge, our IHC results demonstrated for the first time that the presence of HLA-E in the UC should be considered when building the UC as a potential graft source for transplantation in urologic reconstruction. Our observation that there seems to be a paired expression of HLA-ABC and HLA-E at the same regions within the UC tissue is promising despite the lack of HLA-G expression. Normally, HLA-E is known to present peptides derived from the leader sequences of HLA-ABC ,34,38. Oll lysis ,42.In the context of allogeneic graft transplants, mixed results have been reported regarding the effect of HLA-E on transplant outcome and graft survival in clinical and animal studies. HLA-E mismatch between the donor and recipient patients with acute leukemia was found to be associated with a better five-year overall survival and lower graft vs. host disease incidence in the recipient patients . In anotThe UC has been used as allografts in the repair of gastroschisis, foot and ankle wounds, and diabetic foot ulcers ,13,14,15In severe cases of hypospadias, the buccal mucosal graft or preputial skin graft are commonly used for urethral reconstruction . HoweverLimitations exist in the present study. Since the sample size of this study was small, the reported findings should be used as the initial supporting evidence for more extensive research on the clinical implications of the immunological profile of UC allografts. Using a preclinical model, it would be important to investigate whether the localization and relative levels of HLA molecules in different UC regions contribute to differences in the engraftment outcome. Since the present study focused on the semi-quantitative comparison between HLA molecules by histological staining, future quantitative analyses are needed to further confirm the suggested immunological interactions between HLA-ABC and HLA-E. It would be useful to sort hematopoietic progenitor cell subpopulations by their HLA expression using flow cytometry. Furthermore, the differences in their long-term culture-initiating cell (LTC-IC) frequency and generation of more mature precursor cells, such as burst forming unit-erythroid (BFU-E) cells (BFU-E), could be explored in colony and co-culture assays. In conjunction with the findings reported in this study, gaining a deeper understanding of the hematopoietic cell subpopulations in the UC tissue will further elucidate mechanisms behind the UC’s low immunogenicity in allogeneic environments.Overall, we observed a paired localization of HLA-ABC and HLA-E in UC tissue and that the level of HLA-G appears to be lower than HLA-ABC and HLA-E. It is likely that the UC maintains some level of immunosuppression by virtue of the paired expressions of HLA-ABC and HLA-E and their known interactions on immune cell activity. Future attempts to develop tissue grafts out of UC should use the histology protocol we described to characterize and determine the utility of any UC that is to be developed into a future graft.


In this study, we developed a new formulation for oral administration by reacting GA and L-Arg to form salts by co-solvent evaporation and then adding the polymer-solvent Soluplus® with an amphiphilic chemical structure to prepare a solid dispersion GA-SD. The chemical and physical properties of GA-SD were characterized by DLS, TEM, XRD, FT-IR and TG. The anti-inflammatory activity of GA-SD was verified by LPS stimulation of RAW 267.5 cells simulating a cellular inflammation model, TPA-induced ear edema model in mice, and ethanol-induced gastric ulcer model. The results showed that the amide bond and salt formation of GA-SD greatly improved GA solubility. GA-SD effectively improved the anti-inflammatory effect of free GA in vivo and in vitro, and GA-SD had no significant effect on liver and kidney function, no significant tissue toxicity, and good biosafety. In conclusion, GA-SD with L-Arg and Soluplus® is an effective method to improve the solubility and bioavailability of GA. As a safe and effective solid dispersion, it is a promising anti-inflammatory oral formulation and provides some references for other oral drug candidates with low bioavailability.Glycyrrhetinic acid (GA) is an anti-inflammatory drug with potential for development. However, the poor solubility of GA in water leads to extremely low bioavailability, which limits its clinical applications. Solid dispersions have become some of the most effective strategies for improving the solubility of poorly soluble drugs. Soluplus Inflammation is the first protective response in biological systems and can protect against injury caused by harmful stimuli, such as physical insults, chemical insults, or infection ,2. HowevGlycyrrhetinic acid (GA) is a triterpene saponin generated by the hydrolysis of glycyrrhizic acid to remove the sugar and acid chains and is the main active ingredient in licorice. According to the Biopharmaceutical Classification System, GA is a type II drug . GA has ®) is a noncytotoxic amphiphilic solubilizer, absorbent, and penetrant that can be used as a carrier in the formulation of solid dispersions [® allows it to act as a matrix polymer for solid solutions and to increase the solubility of insoluble drugs [® is 6.63 × 10−3 mg/mL [® exceeds the low critical micelle concentration, Soluplus® encapsulates the hydrophobic drug in a hydrophobic core where the carbonyl group in the molecular structure binds to the hydroxyl group of the drug. At the same time, Soluplus® prevents crystal formation by reducing the energy of the particle surface, further improving the stability of the micelles [® can significantly increase the dissolution of BCS II drugs and improve the bioavailability of insoluble drugs [Solid dispersions have become some of the most effective strategies for improving the solubility of poorly soluble drugs ,13. The persions . The bifle drugs . The low−3 mg/mL . When thmicelles ,21. Studle drugs . L-arginle drugs . These ile drugs . It has le drugs .® as a polymer to improve the solubility and bioavailability of free GA and provide a new scheme for the preparation of GA-SD. The effects of GA-SD on the anti-inflammatory activity of free GA were analyzed in an LPS-stimulated cellular inflammation model, a mouse ear edema model and an alcohol-induced gastric ulcer model. The biosafety of GA-SD was also demonstrated. The results showed that the GA-SD solid dispersion system developed in this study is promising for improving the bioavailability and anti-inflammatory effects of free GA. With the improvement of the solubility and utilization of GA, the application scope of GA in the treatment of many inflammatory diseases will be expanded.This study aimed to prepare solid dispersions with L-Arg as an excipient and Soluplus® was purchased from BASF , and L-Arg was purchased from Beijing Aibifan Biotechnology Co. 12-O-tetradecanoylphorbol-13-acetate (TPA) . All other related chemicals and reagents were analytically pure.GA was purchased from Beijing Mairuida Technology Co., Ltd., SoluplusTM, Sydney, Australia) at 37 °C in a 5% CO2 incubator and passaged by gently scraping the adherent cells with a cell scraper at a ratio of 1:6–1:8.The mouse macrophage RAW264.7 cell line was purchased from the Cell Resource Center of Peking Union Medical College Hospital. RAW264.7 cells were cultured in DMEM complete medium  containing 10% fetal bovine serum  were purchased from Beijing Huafukang Biotechnology Co. All animals were kept under stable temperature conditions (20–26 °C) and 12 h light/dark cycles for three days. All experiments were conducted by the regulations of the National Institutes of Health (NIH) and the Beijing Laboratory Animal Ethics Committee.® were prepared in the proportions shown in ® solution was prepared with 50 mL of deionized water, and the sample was redissolved by adding the prepared Soluplus® solution to the rotary dried sample. The hydrophilic L-Arg fraction allowed the GA-L-Arg complex to disperse in water and the hydrophobic GA fraction combined with the hydrophobic structure of Solulplus® to form the GA-L-Arg-Solulplus® ternary complex. The resulting solution was a GA solid dispersion. The samples were freeze-dried to obtain a solid dispersion powder.To prepare GA-SD, the co-solvent evaporation method was used ,26. BrieIn this study, the drug loading capacity of the prepared GA-SD was tested to screen for the optimal reagent ratio for the preparation of solid dispersions. GA solid dispersions prepared under different mass ratios of substances were first taken and dissolved in deionized water, and then the drug loading was detected by HPLC under the following conditions: phase A methanol, phase B 0.2% phosphoric acid, A:B = 90:10, flow rate 1 mL/min; wavelength 250 nm, column temperature 35 °C; each injection volume was 20 µL.® and GA-SD were weighed respectively and configured into a 2 mg/mL solution. All the samples were diluted 3 times with deionized water at a ratio of 1:3 (v/v), and then the average particle size and Zeta Potentials were measured by a nanoparticle size potential analyzer.A Nano-ZS 2000  was used to determine the particle size and Zeta potential. Before measurement, an appropriate amount of SoluplusMorphological observation of GA-SD was performed using a Hitachi-HT7700 (120 kV) transmission electron microscope. Double distilled water was used to dilute the GA-SD powder and ultrasonically disperse it for 30 min. A drop of diluent (approximately 5 μL) was loaded onto a copper mesh  coated with formaldehyde/carbon, and the sample was air-dried before observation.®, L-Arg, a physical mixture of the three, and GA-SD powder were scanned in the range of 4000–400 cm−1 by FT-IR (Thermo Scientific Nicolet iS20).Infrared analysis was performed to identify the formation of complexes by evaluating deviations in peak shape, position, and intensity. A certain amount of GA, Soluplus®, L-Arg, their physical mixture and GA-SD powder were taken and laid flat on a sample table and then analyzed by XRD. The scanning speed was 5°/min, and the scanning range was 5–80°.To observe the state of the GA before and after wrapping, the GA-SD powder was characterized by X-ray crystal diffraction (XRD) (Pert3 Powder). Certain amounts of GA, SoluplusThermogravimetric analysis (TA SDT 650) was employed to evaluate the thermal stability of GA-SD. The samples of about 10 mg were sealed in an aluminum plate and heated from 30 °C to 800 °C with the rate of 10 °C/min under an atmosphere of nitrogen. The weight variation of the samples was recorded in relation to temperature.To investigate the stability of the prepared GA-SD, we took 50 mg of the prepared GA-SD powder and dissolved it in 25 mL of deionized water. The studies were carried out at room temperature for 63 days. The change in GA content in the solid dispersion was measured by HPLC every 7 days. Meanwhile, the GA-SD were stored at 4 °C for 7 months and analyzed by X-ray crystal diffraction to determine the crystallization propensity of GA-SD over time.The GA-SD (500 mg) was respectively added to a 15 mL centrifuge tube containing 2 mL deionized water and then shaken at 37 °C for 72 h. After the shaking, the samples were filtered with a 0.22 filter membrane and diluted with absolute ethanol. Then, the GA was quantified by HPLC  of GA and GA-SD, respectively, for 24 h. Then, CCK-8 solution was added and the optical density (OD) at 450 nm was determined. GA was dissolved in DMSO, and the volume of DMSO used was one-thousandth of the volume of the medium [The safe concentration of cell administration on RAW264.7 cells was assessed by CCK-8 assay. Briefly, cells dispersed evenly in the medium were seeded at a density of 5.0 × 10e medium . The dis5/mL cell suspension, which were inoculated in 96-well cell culture plates with 100 µL of cell suspension per well. They were incubated in a 37 °C, 5% CO2 incubator. When the cells were spread in a monolayer across the bottom of the well, the supernatant was removed, and 100 µL of the serum-free medium was added as the normal group, 100 µL of serum-free medium containing Lipopolysaccharide (LPS) (1 µg/mL) as the model group, and 100 µL of serum-free medium containing a final concentration of 50 µM GA, GA-SD and LPS (1 µg/mL) as the experimental group [RAW264.7 cells in the logarithmic growth phase were collected by trypsin digestion and configured into a 1 × 10al group . The culThe mice were randomly divided into 4 groups, each with 5 animals. These groups included the control group, TPA model group, GA group (20 mg/kg) and GA-SD group (20 mg/kg) . All druTo evaluate the therapeutic effect of GA and GA-SD on ear inflammation, ear tissues were collected from each group of mice, and to evaluate the hepatic and renal toxicity of GA and GA-SD, liver and kidney tissues were collected after perfusion and paraffin sectioning. The ear tissue and liver and kidney tissues were first fixed by immersion in 4% paraformaldehyde with gradient dehydration and paraffin embedding. They were sliced into 4 μm post-thin sections. They were stained with hematoxylin-eosin stain .The sections were observed by optical microscopy, and three visual fields were randomly selected for analysis. Image analysis was performed by a pathologist who did not know the experimental conditions to evaluate the degree of tissue damage.The orbital venous blood of mice in the normal group, the GA experimental group and the GA-SD experimental group were centrifuged at 3000 rpm for 10 min, and the serum was collected. According to the manufacturer’s instructions, the serum total protein (TP), serum albumin (ALB), creatinine (CREA), urea (UREA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured with a commercial kit (CSCN). According to the normal reference values of the biochemical indices of Kunming mice provided by Huaying Biology, the biosafety of GA and GA-SD was evaluated.n = 5) as follows: normal control group; model group; GA group (20 mg/kg) and GA-SD group (20 mg/kg)  . Mice inOne hour after the ulcer induction, animals were anesthetized with isoflurane and euthanized by cervical dislocation. The excised stomach was opened along the greater curvature and rinsed completely with PBS. Then, the stomachs were blotted dry with filter paper, spread out, and photographed. For the determination of gastric lesion area, the gastric ulcer index (GUI) was calculated by analyzing the inner surface of the stomach using Image J software (v1.53c.). The gastric tissues were taken for pathological section analysis after the photoshoot. For each group, the gastric ulcer index was determined using the following equation: The percentage of ulcer preventive index was calculated as follows:After a general examination, gastric tissues were preserved in a 4% paraformaldehyde buffer solution for 24 h and then embedded in paraffin wax. Slices were prepared and stained with hematoxylin and eosin (H&E) for histological evaluation.2O2 to inhibit endogenous peroxidase activity and blocking with 3% BSA for 30 min. After that, the slices were treated with primary antibodies diluted at 1:500 overnight at 4 °C, and then with peroxidase-coupled secondary antibodies for 50 min. Color development was carried out by incubating with 3,3′-diaminobenzidine tetrahydrochloride for 5 min. The results were captured by light microscopy, followed by analysis by Image J software.IHC staining was employed to assess the expression levels of pro-inflammatory cytokines TNF-α and IL-6. For a start, tissue sections were deparaffinized, rehydrated, and then antigen retrieved. Subsequently, the sections were pretreated for 25 min with sodium citrate buffer via heat-mediated antigen retrieval followed by treatment with 3% Ht-test. The significance levels (p ˂ 0.05) were regarded as statistically acceptable.All results are presented as the mean ± standard deviation (SD). Statistical comparison was made via Student’s ® was added, and Soluplus® formed solid dispersions of the solutions via hydrogen bonding or complexation reactions.GA-SDs were prepared using the co-solvent evaporation method. As described in the experimental section, the GA and L-Arg solutions were mixed thoroughly and left for 30 min to form salts. Then, Soluplus® was less than 1:1:2 (w/w/w), the encapsulation efficiency gradually increased to nearly 100%. At this point, we believed that GA-L-Arg was excessive and more Soluplus® provided more loading space, leading to the increase in the encapsulation efficiency. As the Soluplus® content increased, the entrapment efficiency did not increase further but instead decreased slightly, possibly because the excess dissolved Soluplus® seized too many water molecules, resulting in there not being enough water to rehydrate with GA-Arg, making redissolving difficult [As shown in ifficult .® was determined to be 1:1:2.The best mass ratio of GA, L-Arg and Soluplus® and prepared GA-SD were measured by DLS. The result is demonstrated in ® could form micelles spontaneously due to its amphipathic property. The results of zeta potential indicated that Soluplus® micelles were formed with nearly neutral charges. Therefore, the charge of GA-SD should be derived from its loading components. We tested the potential of the GA-Arg complex and found that its charge was −3.4 mV, while GA-SD loaded a large number of complexes, which made the surface charge accumulate, showing the zeta potential was approximately −4.15 mV. An important factor in measuring the performance of nanoparticles is their particle size, which affects the circulation and biodistribution of the drugs [® was 63.17 nm, and the average particle size of GA-SD was 68 nm, which was very close to the particle size of Soluplus®. The DLS results of GA-SD showed a sharp peak, indicating single and narrow particle size distributions. Additionally, the microscopic details of GA-SD were confirmed by TEM , COO– stretching (1420 cm−1), and NH2 deformation (1134 cm−1). Carboxylate anion usually shows an absorption peak at 1650–1550 cm−1 due to strong asymmetric carboxylate stretching [−1) of the carboxyl group in GA suggested the ionization of the carboxyl group [−1 and 1368 cm−1, attributed to the asymmetric (Vas COO−) and symmetric stretching vibration (Vs COO−) of the –COO− group [XRD and FTIR were used to characterize the interaction forms and the structure of the solid dispersion. The XRD results are plotted in lic acid . These ssegments . The infsegments ,37. The retching . The disyl group ,40,41. Tetching 10 cm−1, aO− group . This in® make GA-SD extremely hydrophilic. Therefore, the water solubility of GA was greatly improved.The strong ionic interaction between the GA and L-Arg, the formation of salts and the hydrophilic structure of SoluplusThe TGA scan of GA showed almost a constant weight until 300 °C . GA bega® and its crystallization inhibitory effect [The stability of GA-SD is shown in y effect .−3 mg/mL . The result of the cell viability assay is shown in ®), leading to higher solubility. Moreover, the suitable particle size of GA-SD enhanced the permeability of its cell membrane, which also improved the utilization of the drug by the cells and enhanced the anti-inflammatory effect.LPS stimulation of the mouse peritoneal mononuclear macrophage cell Line 264.7 was used to simulate the inflammatory response in vitro, and the cell supernatant was analyzed for inflammatory factors by ELISA. When cells are stimulated by LPS, the secretion of proinflammatory cytokines, such as IL-1β, IL-6, MCP-1, TNF-α, IL-23, and IL-17A, increases, while the secretion of anti-inflammatory cytokines such as IL-4 and IL-10 decreases. Cytokines, such as IL-1β, TNF-α, and IL-6, are key proinflammatory factors that can mediate inflammation by inducing cellular pro-inflammatory gene expression ,47. IL-1Based on the current anti-inflammatory effect of solid dispersion GA-SD in vitro, this study was conducted to construct a mouse ear edema model by applying TPA to the outer part of the mouse ear, and the anti-inflammatory effect of GA-SD in vivo was evaluated by gavage administration, using free GA in the control group. The experimental scheme A and the® is biocompatible and could be a suitable candidate for orally administered dosage forms [Arg is a unique, non-toxic, and biocompatible biomolecule . Parikh ge forms . GA-SD pThe liver is the main target organ for toxic compounds, while the kidney is a metabolic organ with multiple functions, such as eliminating blood waste and balancing body fluids. In this study, we examined alterations in liver and kidney function biomarkers  in male Kunming mice after oral administration of GA and GA-SD, thus, assessing the effects of GA and GA-SD on liver and kidney function. Increase in liver enzymes is related to the change in cell membrane permeability caused by liver and hepatocyte damage, so the change in liver enzyme content reflects the hepatotoxicity of the drug. Creatinine and urea are important indicators used to reflect the detoxification of the kidneys, by which the status of renal function is judged as normal ,56. The To further evaluate the anti-inflammatory effect of GA-SD, we established an ethanol-induced gastric ulcer model A. One hoThe results of hematoxylin and eosin-stained (H&E) gastric sections further verified the enhanced anti-inflammatory effect of GA-SD D. ExaminGastric mucosa damage activates the inflammatory process, which increases the secretion of inflammatory cytokines, leading to gastric mucosal damage ,59,60. P® with an amphiphilic chemical structure. GA-SD solid dispersions with particle size less than 100 nm, irregular morphology and good stability were successfully prepared. GA-SD has a particle size below 100 nm, which allows GA-SD to avoid rapid clearance by the reticuloendothelial system and increases cytomembrane penetrability. The presence of L-Arg and Soluplus® in the solid dispersion enables GA-SD to have high solubility; therefore, many of the limitations of free GA, such as poor water solubility, low cellular utilization, and low bioavailability, have been solved. The results of anti-inflammatory effect in vitro showed that the immunomodulatory effect of GA-SD was superior to free GA and GA-SD enhanced the anti-inflammatory effect of free GA in vitro. The anti-inflammatory effect of GA-SD in vivo was evaluated by gavage administration in a TPA-induced mouse ear edema model and ethanol-induced gastric ulcer model. The results indicated that GA-SD effectively improved the anti-inflammatory effect of free GA in vivo. Furthermore, GA-SD had no significant effects on liver and kidney functions, there was no significant tissue toxicity and it displayed good biosafety. With the improvement of the solubility and utilization of GA, the application scope of GA in the treatment of many inflammatory diseases will be expanded. As a safe and effective solid dispersion, it is a promising anti-inflammatory oral formulation and may provide some references for other oral drug candidates with low bioavailability.In this study, we used the co-solvent evaporation method to make GA and L-Arg react to form salts and then added the polymer-solvent Soluplus